A poorly understood step in the HIV-1 life cycle is disassembly of the viral core soon after entry into target cells. Studies of this process have been hampered by the high percentage of defective HIV-1 particles and by the lack of available assays for analyzing early post-entry events in infection. In this application, we propose to dissect the mechanism of core disassembly and its role in HIV-1 infection by employing a combination of biochemical and genetic approaches. Using a panel of HIV-1 capsid and matrix mutants, we will identify the viral determinants of core disassembly by isolating cores from mutant virions and quantifying their stability in vitro. Alterations in core stability will be correlated with the ability of mutant viruses to replicate in cell culture and to undergo reverse transcription in target cells and in permeabilized virions. The role of viral core formation in condensation of the viral ribonucleoprotein (vRNP) complex will be determined by isolating vRNP complexes from HIV-1 particles containing unstable cores and from HIV-1 gag mutants arrested in various stages of maturation. Finally, the putative role of producer cell factors in regulating HIV-1 core stability will be investigated by assaying the core stability and infectivity of HIV-1 capsid mutants produced in various T cell lines. These studies will provide fundamental new insights into mechanisms by which HIV-1 initiates its replication program and may reveal new avenues for targeted therapeutic intervention.